A goal of our research is to understand interactions between the immune system and skeletal muscle that are important in regulating muscle repair, growth and regeneration. By better understanding these relationships, therapeutic manipulations of the immune system or its products can be designed to improve muscle growth and regeneration following acute trauma, disuse or chronic disease. Over the last several years, knowledge in this area has advanced substantially, showing that immune cells called M2 macrophages play a major role in muscle growth and regeneration. However, the identity of the specific, M2 macrophage-derived molecules that drive regeneration remains unknown. We propose to test the novel hypothesis that the protein Klotho generated by M2 macrophages is essential for normal growth and regeneration of muscle following injury. In addition, we will develop a novel, cell-based mechanism for targeting Klotho to injured muscle on demand, so that expression of the therapeutic protein is elevated specifically at the location and time of muscle regeneration. We propose to use an acute, muscle-injury model in mice to address the following aims: Aim 1: Test the hypothesis that genetic or pharmacological interventions that modulate Klotho levels affect muscle regeneration. We will manipulate Klotho levels in mice that experience muscle injury by expressing a Klotho transgene, by ablating the Klotho gene, by transplanting bone marrow-derived cells (BMCs) from Klotho transgenic mice and by administering Klotho protein. Mice will be assessed by molecular, physiological and morphological assays to determine the effects of modified Klotho levels on muscle growth and regeneration. Aim 2: Test the hypothesis that Klotho modulates shifts in inflammatory cells from pro-inflammatory M1 macrophages to pro-regenerative M2 macrophages following muscle injury. We will test whether genetic or pharmacological manipulations of Klotho affect macrophage phenotype or function following muscle injury. Aim 3. Test the hypothesis that myeloid cells can be designed to target elevated Klotho expression to injured muscle during regeneration. We will generate a transgenic mouse line in which Klotho expression is driven by a promoter that is most-highly activated in myeloid cells in the context of inflammation and then assay whether transplanting BMCs from those mice promotes growth and regeneration of injured muscle in the recipients. We anticipate that these findings will establish the importance of Klotho in driving growth and regeneration of injured muscle. We also believe that this work can provide a basis for a new, cell-based delivery of therapeutic molecules to sites of injury and repair that may have significance beyond the treatment of muscle injuries.